1. Field of the Invention
The present invention relates to apparatus and method for mass spectrometry for quickly performing qualitative and quantitative analyses of quite complex sample mixtures typified by peptide mixtures obtained by enzymatic digestion of proteins.
2. Description of Related Art
In recent years, proteins have been identified by comparing mass spectra of proteins or peptides obtained by enzymatic digestion of proteins against a database or library of information about known amino acid sequences of proteins (molecular structures). Peptide mixtures obtained by enzymatically digesting and fractionating proteins include tens of kinds of peptides. It is regarded as important that accurate mass spectral information about at least several kinds of peptides be obtained, in order to precisely identify peptides.
Liquid chromatography is widely used to separate and analyze various components of samples/solutions, such as peptide mixtures. Such a liquid chromatograph is made up of a sample-separating portion and a chromatogram-measuring portion. The sample-separating portion includes a pump, an injector, and a column. A mixture sample consisting of plural components is separated into the components by the sample-separating portion. The process of elution is measured by a mass spectrometer. A mass spectrum of each isolated component can be obtained. See Japanese Patent Laid-Open No. 2001-50945 and Japanese Patent Laid-Open No. H1-118763.
However, with the existing liquid chromatograph, it is often quite difficult to completely separate plural components in a single chromatographic run. Especially, in order to completely isolate naturally occurring samples, such as digestive products enzymatically derived from proteins, it is necessary to discuss the separation conditions of the liquid chromatograph according to each sample, thus requiring much labor and time.
In addition, even if a lot of labor and time are spent, components of interest cannot be isolated in many cases. It has been quite difficult to obtain a mass spectrum of each component using an easy method.
As an example, FIG. 1 shows an enlargement of a part of a mass chromatogram of a peptide mixture obtained by enzymatically digesting fetal bovine serum albumin with trypsin. The solid line indicates the total ion chromatogram. The broken line indicates a chromatogram obtained at m/z=1479.43. The dot-and-dash line indicates a chromatogram obtained at m/z=966.474.
As is obvious from the diagram, with respect to a peak with 36.65 min, at least two different components are eluted. Any region of the peaks which is sufficiently isolated to produce a pure spectrum hardly exists.
In this case, with the prior art technique, any one of the following methods has been used to obtain a separate mass spectrum of each component:
(1) The components are separated after rediscussing the separation conditions.
(2) MS/MS is used.
However, a lot of time, labor, and skillfulness are required to rediscuss the separation conditions. Furthermore, in the case of complex mixtures, such as protein decomposition products, it is customary that not all components can be separated in one chromatographic step.
In the method using MS/MS, substances of interest are selectively isolated using a mass spectrometer after chromatographic separation. Then, a mass spectrum is obtained and so it is possible to quickly solve the foregoing problem. However, a MS/MS instrument is generally expensive. Furthermore, complex manipulations are required. Consequently, there is a demand for an easy and inexpensive method.